Furthermore, the binding capacity was 36.48 ± 0.09 mg/g, similar to CBP. However, both CBP and CBP-H could promote osteogenic activity, the activity of CBP-H was 127.147%, lower than CBP (121.777%). While it had the same ability to promote intracellular calcium concentration, CBP-H could upregulate 150.12% calcium ions into the intracellular, and the rate of the rise of CBP was 158.91%, further highlighting the potential of His residues for binding calcium and treating osteoporosis.Histidine (His) carries a unique heteroaromatic imidazole side chain and plays an irreplaceable role in peptides and proteins. With the current study, we aimed to determine the characteristics and functional activities of the bone density of soy peptide–calcium complexes in which a His residue was replaced by Leu (CBP-H). Soybean peptide (CBP-H) was chemically synthesized, the binding mechanism between CBP-H and calcium ions in combination was determined with bioinformatics and spectroscopy analysis, and the difference between CBP and CBP-H was investigated. Finally, we analyzed the effect of CBP and CBP-H on osteoblasts in vitro. The results showed that CBP-H could bind to calcium ions, and the calcium coordinated with the carboxyl groups of Asp and Glu in the peptide. The nitrogen atoms of the amino group and the oxygen atoms of the carboxyl group in CBP-H significantly contributed to the coordination with Ca Peptides are critical bio-molecules of the human body. They act in living systems, immune regulation, and metabolism. Traditionally, naturally occurring peptides have long been regarded as therapeutic agents and used as a source of new drug development for a long time. For example, casein phosphor-peptides (CPPs), duck egg peptide (VSEE), lactoferrin-derived peptide (PKSETKNLL), soy peptide (DEDEQIPSLPPR), and collagen-binding motif peptide  have been proven to enter cells to promote bone proliferation and differentiation ,2. Howev2+ and Ca2+ and other amino acids through cation–π interactions, π–π stacking interaction, hydrogen–π interaction, and coordinate bond interaction [2+ and Ca2+ was greatly higher than Na+ and K+, and could also bind to host proteins by coordinate bonds [Among the 20 natural amino acids, histidine (His) plays an important role in the peptide bioactivities . The muleraction . The niteraction . The binte bonds . In addite bonds . In compte bonds . Recentlte bonds . HoweverTo further investigate the binding mechanism of peptide and calcium, His was replaced with Leu in CBP (CBP-H) (DEDEQIPSLPPR), UV spectroscopy, Fourier infrared spectroscopy (FTIR), X-ray diffraction spectrum (X-ray), isothermal titration calorimetry (ITC), and molecular docking were used to analyze the interaction between CBP-H and calcium. Thermogravimetric analysis-differential scanning calorimetry (TG-DSC) and Caco-2 cells were used to analyze the stability of CBP-H–Ca. Furthermore, the effect of CBP-H on the cell viability of MC3T3-E1 cells was detected. Finally, the effect of CBP-H on bone mass in vivo by the zebrafish osteoporosis model was detected in this study.2O) and solution B (0.1% formic acid in acetonitrile) using a gradient of 5–95% B. The flow rate was set at 600 mL/min for 78 min. The peptide sequences were detected using a Q-Exactive mass spectrometer after the separation.Polypeptide CBP (DEDEQIPSHPPR), in which a His residue is replaced by Leu (CBP-H), was synthesized by Nanjing Peptide Industry Biotechnology . High-performance liquid chromatography (HPLC) analysis was carried out to determine the purity (98%) of CBP-H, and ESI mass spectrometry was used to confirm the peptide structure. An Agilent MALDI-TOF-MS/MS spectrometer was used to identify the eluted fractions individually . An Agilent 1100 HPLC system, electrospray ionization (ESI) interface, and an Agilent 1100 HPLC system were installed, together with the spectrometer. An automatic sampler was used to inject the sample into a C18 trap column , followed by injection into a C18 column . Elution was conducted with the aid of solution A  was performed according to the method of Cui with slight modification . First, The content of Ca in the CBP-H–calcium complexes was determined by inductively coupled-atomic emission spectrometry (ICP-AES). The detection conditions were as follows: generator power: 1.4 KW, cooling gas flow: 12 L/min, auxiliary gas flow: 0.8 L/min, carrying gas flow: 0.8 L/min.2 solution (50 mM) was put in the ITC syringe. During the measurements, 20 drops of CaCl2 solution were injected into the sample cell. The titration parameter was configured to inject CaCl2 every 5 min with volumes of 2.5 µL. Nano Analyze software  was applied to evaluate the raw data based on data fitting [Isothermal titration calorimetry measured the thermodynamic parameters of CBP-H binding with calcium. Both the CBP-H and the CBP-H–Ca were dissolved in Tris-HCl buffer solution to a concentration of 2.5 mM. The sample was degassed, followed by filtration through a 0.22 µm filter. A total of 2.5 mM of CBP-H was loaded into the ITC cell, and the CaCl fitting .2, which also dissolved in MOPS solution, was continuously shaken at 50 °C for 1 h to obtain a peptide–calcium chelate and the UV spectra were recorded.The ultraviolet spectra of the CBP-H and CBP-H–Ca complexes were monitored over the wavelength range from 190 nm to 400 nm with an ultraviolet spectrophotometer . For the determinations, 0.02 mg/mL of peptide was dissolved in MOPS solution (pH = 8.0). After that, 0.2 mM, 0.4 mM, 0.6 mM, and 0.8 mM of CaCl−1 at a resolution of 4 cm−1, over 32 consecutive scans. In advance of analysis, 3 mg of lyophilized powdered samples and 200 mg of dry KBr were adequately mixed and tableted at 30 MPa [The FTIR spectra were recorded using an infrared spectrophotometer  in the range of 4000–400 cmX-ray diffraction (XRD) analysis of lyophilized samples was carried out using a diffractometer  equipped with CuKα radiation. The date of diffractograms was recorded for the angle (2θ) ranging from 5 to 85° at a scanning rate of 4°/min, and operating at 40 kV and 40 mA.3. Gastric digestion was performed by incubating the CBP–calcium complex or CaCl2  at a pepsin/substrate ratio of 1:100 (w/w) in a water bath shaker . During the gastric step, the pepsin at 1:25 (w/w) of substrates was added, following by adjusting the pH to 7.5. After that, samples were removed after deactivating trypsin by thermal denaturation for 5 min. Then, the obtained samples were lyophilized and stored at −20 °C for the sake of subsequent studies.The simulation of gastrointestinal digestion was carried out following the method described before with some modifications . The gasThe thermal decomposition of the peptide–calcium chelate was characterized by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) . The lyophilized samples of about 5 mg were weighed and heated from 40 °C to 600 °C, the programmed heating rate was 20 °C/min, and nitrogen air at 30 mL/min.−1 streptomycin, and 100 U mL−1 penicillin, and were incubated at 37 °C in a humidified 5% CO2 incubator. Transepithelial transport through the Caco-2 single cell layer was performed in accordance with Wang et al.’s method [The human intestinal Caco-2 cell line has been extensively used as an intestinal barrier model. These cells were cultured in high-glucose Dulbecco’s Eagle’s medium (DMEM), which contained 10% fetal bovine serum (FBS), 100 μg mLs method . The Han3 cells/well, and incubated at 37 °C (in 5% CO2 and 90% humidity) for 24 h. Then, the media were replaced by CBP at concentrations of 0, 0.7, 7, and 70 μM. After a 72 h incubation period, the cells were treated with 10 μL MTT solution (5 mg/mL in PBS) and incubated at 37 °C for 3 h. After that, the medium was removed and 150 μL DMSO was added to each well followed by shaking the 96-well plates for 10 min. Absorption values of each well were measured by a microplate reader  at 570 nm.The influence of the CBP-H on MC3T3-E1 cell osteoblast proliferation was quantified by using the MTT assay, as previously described by Zhe et al. . Briefly2+ concentration was detected by a flow cytometer, according to Zeng et al. s’ method with slight modifications [5 per well was cultured in a 6-well plate. After being treated with CBP-H for 24 h, cells were collected, and 2.5 μ mol/L FLou-3/AM was added and incubated for 1 h. The average fluorescence intensity of cells in each group was detected at a 488 nm excitation wavelength and a 530 nm emission wavelength.The influence of the CBP-H on intracellular Caications . Almost p < 0.05) .All diagrams were drawn using Origin 8.6 software  and Primase 9.0 software . All indicators were determined at least in triplicate, and the data are presented as the mean value ± SE. The significant differences were analyzed and evaluated using Duncan in SPSS 19.0 software  were selected to analyze the interaction between CBP-H and calcium. As shown in 2+ ions through a combination: One Ca2+ ion is sandwiched between two acidic amino acids, and the other calcium ion interacts with Asp-1. As a result, Asp-1 might bind to calcium ions in either a bidentate or unidentate mode, under different circumstances. This binding phenomenon stabilizes the conformation of the N-terminus and C-terminus of the peptide.Intermolecular interaction analysis of the CBP-H–calcium complex during the last frames from the 100 ns MD simulation was performed to compare and clarify the interaction modes and binding type between the CBP-H and calcium. As shown in To clarify the calcium-binding capacity of CBP-H, Nanjing Peptide Biotechnology Corporation. Ltd.  synthesized the peptide CBP-H using a solid-phase procedure. High performance liquid chromatography (HPLC) analysis was performed to determine the purity (98%) of CBP-H , and ESI2). First, the reference baseline was determined by titrating Tris-HCl buffer (pH = 8.0) with CaCl2, followed by 2.5 mM CBP-H solution, and 50 mM CaCl2 solution was added to prevent the reaction between the peptides and calcium ions from being incomplete. As shown in 3 M−1, respectively. This result demonstrates that CBP-H possesses at least one functional calcium-binding location. However, the CBP-H sequence had fewer calcium-binding sites than the CBP, in which the reaction stoichiometry (n) was 1.42 ± 0.04 and (5.23 ± 0.32) ×103 M−1, as reported in our previous study.In this research, ITC was used to explore the binding amount of calcium to CBP-H. 2) create polarization change.The UV and FTIR spectra revealed the binding site of the CBP-H–Ca complex. CBP-H showed a strong absorption peak at 200 nm. With an increasing concentration of calcium ions, the absorption intensity decreased from 1.218 to 1.109, with a slight red shift of the maximum absorption wavelength, indicating that a new substance was generated . When th−1 to 3408.69 cm−1, which may be caused by the interaction of –NH2 with Ca2+, resulting in N–H stretching and the replacement of the hydrogen bonding with N–Ca. Moreover, the 1449.90 cm−1 peaks of CBP-H, which resulted from the symmetric stretching mode of C=O, also shifted to a lower frequency (1446.10 cm−1) in the CBP-H–Ca FTIR spectrum. Furthermore, in the fingerprint region, characteristic absorption peaks of the –C–O bond were observed at 1139.74 cm−1 and produced a blue shift to 1203.16 cm−1, respectively, in the CBP-H–Ca spectrum. In summary, the carboxyl group oxygen atoms and amino group nitrogen atoms in CBP-H play a crucial role in the coordination with Ca2+ to form the CBP-H–Ca chelate, which is consistent with previous studies [To further evaluate the development of the CBP-H–calcium chelate, the FTIR spectra of the CBP-H and the CBP-H–Ca are illustrated in The conformational change of CBP-H–Ca is one of the factors affecting the binding strength between calcium and CBP-H. The crystal change was analyzed by XRD. As The change in peptide structure will affect its thermal stability and digestibility. Generally, TG-DSC is used to analyze the thermal stability of different components. As shown in m/z values of the digests was decreased from 710.8283 to 645.2048, and 710.8012 to 581.2893, individually.To investigate the stability of CBP-H and clarify whether the peptide–calcium complex was completely decomposed during simulated gastrointestinal digestion, LC-MS/MS was used to analyze the CBP, CBP-H, CBP–calcium chelate, CBP-H–calcium chelate, and its simulated trypsin digests after Caco-2 absorption. Peptides can exert functional activity after entering cells through endocytosis. In the previous experiments, we found that CBP could stimulate osteoblast proliferation. To investigate the effect of CBP-H on osteoporosis, we tested the impact of CBP-H on the MC3T3-E1 cell proliferation. As shown in As the second messenger, calcium plays a crucial role in the process of cell differentiation. In previous studies, it was found that CBP can promote intracellular calcium concentration and thus promote the formation of new bone . To furt2. Meanwhile, the Asp and Glu in the carboxyl groups of CBP-H were coordinated with calcium. Additionally, there was no significant difference binding capacity between CBP and CBP-H, but the osteogenic activity of CBP-H was lower than CBP. In summary, His residues can affect the structure of peptides and their functional activity, but play a lesser role than Asp and Glu in the interaction with calcium.The amino acid composition of peptides affects the binding activity with calcium. In our previous study, we purified one soybean peptide CBP (DEDEQIPSHPPR) from soy yogurt that had a high ability to bind calcium and promote osteoblast differentiation. Interestingly, the current published literature has pointed out that histidine residues play a crucial role in peptide function due to its special structure. To assess the roles of His residues in peptides to bind with calcium, we first replaced His by Leu in the peptide CBP, named CBP-H, and investigated the interaction between CBP-H and calcium using molecular docking analysis and spectroscopic methods. Caco-2 cell mode was used to analyze the stability during tryptic digestion and explored the roles of CBP-H in the inhibition of osteoporosis by MC3T3-E1 cells. We found that CBP-H indeed exhibited calcium-chelating capacity and the potential to function as a co-factor in preventing osteoporosis. The nitrogen atoms of the amino group and the oxygen atoms of the carboxyl group in CBP-H significantly contribute to the coordination with CaComputer simulation could be utilized to seek active small molecules that fit the binding sites of the receptor ,30. In tTo verify the reliability of results regarding the binding mode and binding site between CBP-H and calcium, we conducted computer stimulations. Spectroscopic methods were used to analyze the binding mode and binding sites including ITC, UV, FTIR, and XRD. Isothermal titration calorimetry was always used to measure reversible reactions between bio-molecules. This methodology provided the affinity constant, stoichiometry, enthalpy, and entropy of the biomolecular interactions. According to the results of ITC, we found that the CBP-H sequence had numerous calcium-binding sites, but lower than CBP. This is consistent with the computer’s prediction. Moreover, the FTIR showed that the CBP-H can bind calcium by binding carboxy oxygen and amino nitrogen. However, the conformational analysis by XRD revealed that the diffraction peak decreased and there was a sharp diffraction peak after changing the amino acid of His in CBP. It has been reported that the imidazole ring of histidine-containing peptides plays an essential role in metal ion binding ,32. The 2+ ions. However, Glu (E) in the CBP chain was not cut-off. This result is consistent with the results of previous studies, showing that dipeptidyl peptidase IV can create hydrogen bonds with sea cucumber ovum-produced heptapeptides and egg white-simulated digestion products, which can suppress the functioning of dipeptidyl peptidase IV via a charge and π–π interaction mechanismThe thermal stability and acid-resisting ability of peptide–calcium complexes affect the bioavailability of calcium. We analyzed the stability of CBP-H using TG-DSC. The results showed that CBP-H had better pH resistance, but lower acid-resisting ability than CBP, implying that His plays a vital role in the conformation of the peptide–calcium complex. To distinguish the stability difference between CBP and CBP-H, the complex and its simulated gastrointestinal digests from Caco-2 cells were evaluated by LC/MS. The initial CBP and CBP-H were also identified in digests, and new other sequences were also identified from the digests. Generally, Leu, as a hydrophobic amino acid, is regarded as the hydrophobic site, but CBP-H was not cut-off at Leu, which is consistent with Cui’s reported data . AlthougThe functional activity changes of peptides could reflect the effect of His modification on peptides. We analyzed the osteogenic activity of CBP-H and found that CBP-H could promote the proliferation of osteoblasts in a dose-dependent manner. However, the inhibited rate of CBP-H was lower than CBP, which may be due to His being able to produce an affinity with the head of the cell phospholipid molecule, improving the binding with membrane receptors and thus promoting osteogenic activity.2+, as a messenger in the cells, plays an important role in many physiological processes such as initiating, regulating, and promoting osteoblast proliferation. According to the MTT experiment, we found that both CBP and CBP-H have potential osteogenic activity. To investigate the effect of CBP-H on osteogenic activity, we detected the intracellular calcium concentration. CBP-H can upregulate the intracellular calcium concentration, and the increase ratio was similar to CBP, suggesting that both CBP and CBP-H may promote osteogenic activity by binding with receptor protein in the cell surface and entering the cytoplasm through endocytosis.Finally, Ca2+. However, further studies are warranted to focus on the mechanisms underlying the anti-osteoporosis effects of amino composition.His residence in CBP is critical for binding calcium. The calcium ion can coordinate with the Asp and Glu of carboxyl groups in the peptide. The nitrogen atoms of the amino group and the oxygen atoms of the carboxyl group in CBP-H significantly contribute to the coordination with Ca


There was no significant difference in body weight change between insulin users and non-insulin users at 12 months . The proportion of responders (decrease of HbA1C > 1%) in insulin users was much higher than that in non-insulin users . In insulin users, those who had increased insulin dosage at 12 months had significantly less HbA1C reduction than that of non-increased patients . GLP-1 RAs provide superior glucose-lowering effects in insulin-treated patients compared with non-insulin-treated patients with T2D without significant differences in body weight decrease.Glucagon-like peptide 1 receptor agonist (GLP-1 RA) is a potent antidiabetic agent with cardiorenal and weight-losing benefits in patients with type 2 diabetes (T2D). The combination of GLP-1 RA with basal insulin has been suggested in several clinical studies as a useful treatment for intensifying insulin therapy in T2D. However, there has been no real-world evidence study comparing the glycemic effects of GLP-1 RAs add-on to background treatment with and without insulin. A retrospective study was performed in 358 patients with T2D who initiated liraglutide or dulaglutide. Among them, 147 patients were prior and concurrent insulin users, and 211 patients were non-insulin users. After 12 months of GLP-1 RA treatment, the changes in hemoglobin A1c (HbA1C) and body weight were evaluated. The effectiveness of GLP-1 RAs on HbA1C reduction was greater in insulin users than non-insulin users at 12 months (−1.17% vs. −0.76%;  Type 2 diabetes mellitus (T2D) has become an important health issue worldwide since the number of people with diabetes is increasing gradually . The totIn recent years, many new antidiabetic agents have been developed targeting different metabolic pathways involved in T2D . CurrentDespite not being recommended, insulin is still commonly prescribed as the first injectable therapy prior to GLP-1 RAs for inadequately controlled diabetes in real-world practice owing to its cost-effectiveness. Although the positive effects of GLP-1 RAs add-on therapy to insulin have been demonstrated in many studies ,14,15,16p < 0.001), had longer diabetes duration , and had higher HbA1C , whereas body mass index (BMI) was lower . The proportion of patients with an eGFR < 45 mL/min/1.73 m2 in the insulin group was higher than in the non-insulin group . In addition, the insulin group was treated less often with metformin  and insulin secretagogues  than the non-insulin group.We enrolled 358 consecutive patients in this study. The patients were divided into two groups based on the presence or absence of prior and concurrent insulin treatment. Among them, 147 patients were insulin users, and 211 patients were non-insulin users. The baseline characteristics of the study subjects are shown in p = 0.018) (p = 0.287). When calculating patients treated with liraglutide (n = 217) or dulaglutide (n = 141) separately, we found a similar trend in both insulin users and non-insulin users of these two GLP-1 RAs. This justifies combining liraglutide and dulaglutide as “GLP-1 RA” in our study. In contrast, in patients who initiated insulin during the same period , the HbA1C improved significantly after insulin treatment , but was accompanied with increased body weight . These results are compatible with current knowledge that insulin has glucose-lowering effects at the expense of gaining weight.As for glycemic control, we found that GLP-1 RAs had a greater effect on HbA1C reductions in insulin users than non-insulin users at 12 months  A. This d= 0.018) B. This wp = 0.04). However, there were no differences in HbA1C change  (p = 0.689)  A and bod= 0.689) B betweenn = 63, 43%) was defined by an insulin-dosage increase of ≥ 0 IU at the 12th month and all the others (with unchanged or decreased dosage) were designated as the non-increased group . We found both increased and non-increased groups had comparable fasting plasma glucose, HbA1C, diabetes duration, and body weight at baseline. As a whole, the total daily insulin dose (TDD) was not changed in insulin-treated subjects . Interestingly, we noted that the increased group had a significantly less HbA1C reduction than that of the non-increased group . However, there was no significant difference in body weight change between the increased group and the non-increased group .Finally, to explore the potential effect of changes in insulin dosage on glycemic and body weight in insulin users, we divided the study subjects by the changes in insulin dosage at the 12th month. The increased dosage group (p = NS). No gallbladder disease event occurred in all of the subjects. We also observed that the urinary albumin-to-creatinine ratio (UACR) decreased significantly in both “insulin use”  and “non-insulin use” groups , indicating renal benefits of GLP-1 RAs with/without insulin.During the study period, one patient in the “insulin use” and two patients in the “non-insulin use” group developed acute coronary syndrome  than the non-increased group. This finding indicates that patients in the increased group did not respond well to GLP-1 RA therapy so insulin doses were up-titrated. Our findings are consistent with a meta-analysis of randomized controlled trials, revealing that patients with a good response to GLP-1 RA do not exceed 50–60% . RegardiIn our study, the significantly decreased UCAR in GLP-1 Ras-treated patients (with or without insulin) confirmed the renal benefits of GLP-1 RAs. Only three patients developed cardiovascular events during the observation period, which is suggestive of the cardiovascular safety of GLP-1 RAs.There are some limitations in this work. First, as a real-world retrospective study, some baseline characteristics were not comparable between the two study groups. Therefore, we should be careful in interpreting the results. However, after adjustment for covariates, we were still able to demonstrate the significant difference in glycemic control between the groups. Second, since this was a single-center study and the sample size was relatively small, a larger sample could be more readily generalized to the population in the future. Third, only two GLP-1 RAs were analyzed in this study; it remains unclear whether this is a class effect so that newer GLP-1 RAs, such as semaglutide, in either injectable or oral form, have a similar effect. Furthermore, adherence data, which is an important factor affecting the treatment results, are not available in our study. With regard to this issue, nearly 90% of the patients have been enrolled in the structured and integrated “Diabetes Shared Care Program” provided by physicians and certified diabetes educators to ensure compliance. Finally, the findings in our study may not be generalized to other ethnicities since East Asian T2D patients have distinct characteristics, such as early loss of beta-cell function with reduced insulin secretion, compared with Caucasians .The present study was designed as a retrospective observational study. The study protocol was approved by the Human Experiment and Ethics Committee of National Cheng Kung University Hospital (NCKUH)  . Consecutive patients with T2D aged >20 years old who started GLP-1 RA (liraglutide or dulaglutide) between 1 September 2013, and 31 December 2019 (index date) and regularly followed up at the endocrinology out-patient clinic of NCKUH were enrolled. In addition, we retrospectively collected 20 patients with diabetes that initiated insulin during the same period for comparison of the glycemic and weight effects.We excluded the subjects with: 1. a history of pancreatitis; 2. acute infections; 3. active malignant diseases; 4. pregnancy; and 5. who have taken steroids for over seven days. Those who did not follow up within 12 months after GLP-1 RA treatment were also excluded. 2), calculated as weight (in kilograms) divided by body height (in meters) squared [We collected anthropometric parameters, including body height (to the nearest 0.1 cm), body weight (to the nearest 0.1 kg), and BMI (in kg/m squared .2). Events of severe hypoglycemia, defined as a severe event where assistance for the treatment of hypoglycemia is needed [All of the patients fasted overnight for at least 12 h before blood sampling. The fasting plasma glucose, creatinine, alanine aminotransferase, and lipid profiles were measured with an autoanalyzer  at the central laboratory of NCKUH. HbA1c was determined using Tosoh Automated Glycohemoglobin Analyzer . Urinary protein excretion was assessed by urine albumin/creatinine ratio. The modification of diet in the renal disease equation was used to calculate the estimated glomerular filtration rate  with a ratio of non-insulin to insulin groups of 1.5, we would need 219/131 patients in the non-insulin/insulin group to achieve a power of 80% and a level of significance of 5%.t-tests were used, respectively. We considered a p-value of less than 0.05 as statistically significant.For the data analysis, the Statistical Package for the Social Sciences  was used. Clinical characteristics were expressed as mean ± standard deviation (SD) for continuous variables and as percentages for categorical variables. To analyze the differences in categorical and continuous variables between groups, Chi-square and Student’s Our study indicates that GLP-1 RAs can be preferentially combined with basal insulin. GLP-1 RAs provide superior glucose-lowering effects in insulin-treated patients compared with non-insulin-treated patients with T2D, without significant differences in body weight decrease. Therefore, we suggest GLP-1 RAs as a good choice for those who have not achieved glycemic goals even after initiating insulin.


There are three objectives in this study; 1) to measure the live weight of pre-and post-weaning Saanen kids; 2) to determine the growth curves between males and females pre-and post-weaning Saanen kids; and 3) to analyze the body condition score (BCS) between male and female pre-and post-weaning kids.n = 5 male and n = 5 female) and 20 post-weaning Saanen kids  were employed in this investigation. Pre-and post-weaning kids’ live weights were assessed weekly on a weighing scale, and BCS was calculated based on their body frame. In the data analysis, the two-sample t-test with Minitab Software was utilized.This study included pre-weaning kids aged 1 month, as well as post-weaning kids aged 3–6 months. 10 pre-weaning purebred Saanen kids  than the female (75%). Meanwhile, the BCS of pre-weaning Saanen kids grew from week 1 to week 6. It is critical to account for the development of muscle mass while still evaluating the fat cover to determine whether the kids are maintaining an adequate BCS. However, the live weight of post-weaning kids was inconsequential because they were still in the growing phase. As a result, from the 1st to 6th week, post-weaning kids’ body weight and BCS increased as their growth progressed. After 6 weeks of sampling, females had a higher percentage of live weight than males. This is because the kids raised on the farm do not have complete control over the environmental effects. Over 6 weeks of sampling, female post-weaning Saanen kids grew a slightly higher percentage of live weight (88%) than males (85%).p > 0.05). In conclusion, the live weight changes of Saanen kids during the weaning stages are independent of the BCS.This study conducted a direct assessment study, which monitored and determined the live weight and BCS of pre-and post-weaning Saanen kids. Pre-weaning kids’ average values of live weight were calculated as insignificant at the age of 1 month. The mean live weight is most affected by milk consumption from its mothers, the management of the farm, and the environment. For the post-weaning Saanen kids, the females have a slightly higher average live weight gained for 6 weeks than the males ( Commercial dairy goat farms have been established in numerous Southeast Asian countries over the past couple of decades because of the rising consumer demand for goat milk . SmallhoSaanen kids typically weigh between 2 and 4.01 kg at birth, and they can gain up to 12.9 kg –12. BodySmallholders are starting to open more dairy goat farms in Malaysia because of the rising demand for animal products. However, this is insufficient to meet demand, and Malaysia must import goat products, mainly milk and meat, from other countries . One of The weaning stages in this study encompass pre-and post-weaning on a local Saanen dairy goat farm. On this farm, Saanen kids are frequently underweight, which causes the farmer great concern. Previous research has shown that gender can influence growth parameters because of the positive interaction between male gonadal cells and growth hormones, which are linked to growth performance . AccordiIn contrast to other land-dwelling mammals, goat growth and development follow a similar pattern and are influenced by both external and internal climate influences. Saanen kids have varying live weights before and after weaning, which affects how they wean . The groThe study is most important to lessen and achieve non-biased effects on the BCS determined. Thus, it helps the farmers to enhance the quality of their production of Saanen kids on a local dairy goat farm. The results are important to enhance the production of Saanen kids to grow healthy. There are three objectives in this study: to measure the weekly live weight of male and female pre-and post-weaning Saanen kids; to evaluate the BCS of pre-and post-weaning Saanen kids; and to determine the growth curves between males and females of pre-and post-weaning Saanen kids on a dairy goat farm.The sampling methodology and experimental protocols employed in this work were authorized by the UniSZA Animal and Plant Research Ethics Committee (reference number: UAPREC/007/032).The study was conducted on the local dairy farm, Besut District, Terengganu. The geographical location is between 5 38’N and 102˚28’E. The average annual temperature in Besut is 26.9°C. Besut has a rainy season in January, June, July, August, September, October, November, and December.Brachiaria humidicola in the afternoon. Ten Saanen kids (n = 10), 5 males and 5 females pre-weaning, and 20 Saanen kids (n = 20), 10 males and 10 females post-weaning, were used in this study. The live weight of each of the pre-and post-weaning Saanen kids was taken every Tuesday morning until week 6.Pre-weaning Saanen kids aged 1 month and post-weaning kids aged 3 to 6 months were employed for this study. These animals were selected based on the date of birth recorded in animal management records. The pre-and post-weaning kids were placed in Saanen kids’ pens in an intensive housing system where they are limited to grazing. Post-weaning kids were fed twice per day, with pellets in the morning and fresh Pre-weaning Saanen kids were marked on their heads, while post-weaning Saanen kids were marked on their backs. This allowed us to differentiate the samples from other Saanen kids using marking crayons and sex identified. There were 10 pre-weaning  and there were 20  marked in the same pan. The kids were selected and restrained carefully before being weighed. When it came to weight, one end of the rope was tied to each kid’s shoulders, and the other end was tucked under their bellies. A weighing scale with a 100% sensitive balance scale was used to weigh the kids. The kids were weighed once weekly using the same weighing scale upon the mark. The live weight of the pre-and post-weaning Saanen kids was recorded. Thus, an analysis of the performance of growth was recorded. The statistical analysis was determined after weighing the pre-weaning and post-weaning kids.t-test in Minitab Software . The t-test was done to present the average of live weight ± SD and average of BCS ± SD along with the level of significance p < 0.05.The effects of gender on the growth performances of pre- and post-weaning Saanen kids’ were analyzed using a two-sample For this study, 10 pre-weaning kids  of the same age were used. The pre-weaning Saanen kids’ live weights and growth patterns were shown in p > 0.05) over the weeks. Pre-weaning kids’ average live weights were highest at week 6, when they were 9.00 ± 1.41 and 8.80 ± 1.10 kg, respectively. As a result, pre-weaning kids’ body weight varies from the 1st to 6th week as their growth rate increases. The average live weight of females, however, remained the same in weeks 5 and 6. Overall, both males and females exhibit a consistent increase in live weight. It appears that both males and females had the same average live weight in weeks 3 and 4. The pre-weaning kids were in a stage of steady growth.p > 0.05).p > 0.05) over the weeks. The male and female pre-weaning kids’ highest mean BCS values, 3.40 ± 0.89 and 3.60 ± 0.55, respectively, were discovered at week 6. Because they were in the growth phase, pre-weaning kids develop body fat quickly as they transition to post-weaning. When looking at the kids’ fat covers to see if they have a good BCS, it is important to consider the increase in muscle mass as the BCS goes up by weeks.Pre-weaning kids’ live weight was revealed to be insignificant in a previous study  because Portolano et al.  also repAccording to a previous study , the posLive weight is one of the crucial criteria for animal selection . Live weDue to their growth stage, pre-weaning kids’ BCS is challenging to assess. Thus, the results gathered most were BCS 2 and 3, as the pre-weaning kids have normal BCS and are undergoing normal growth performances. There was no paper reporting the BCS in pre-weaning kids because they were still in a growing phase. The BCS can, however, be used to find out if the milk the babies drank before they were weaned was enough for them.Twenty post-weaning Saanen kids aged 3–6 months old were employed for this study. The live weight values represent the respective growth rates of selected post-weaning Saanen kids at a local dairy goat farm  and 6.p > 0.05) over the weeks. At week 6, male and female post-weaning kids had the greatest average live weights, 17.85 ± 3.36 and 17.55 ± 2.25 kg, respectively .According to the current findings, which are shown in For a 6-week sampling period, the female kids in According to The live weight and BCS of pre- and post-weaning Saanen kids were monitored and determined as part of this direct assessment study. At the age of 1 month, the average live weight of pre-weaning kids was determined to be inconsequential. The environment, farm management, and milk consumption from mothers all have an impact on the mean live weight. The pre-weaning Saanen kids’ growth would be impacted by the gender differences in live weight and BCS body composition. Comparison to the male post-weaning kids confirms the fact that the female Saanen kids have a somewhat larger average live weight growth over the period of 6 weeks. Overall, the live weight of the kids is not related to the BCS.


To determine the seroprevalence of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) antibody status amongst healthcare workers (HCWs) working through the first wave of the Coronavirus (COVID-19) pandemic in 2020. To examine the association of seroprevalence and self-reported COVID-19 symptoms with occupation, sex, and ethnicity; and how these factors were associated with physical and mental wellbeing.Single-centre cohort study.Large public hospital in the United Kingdom.All HCWs who had been tested for anti-SARS-CoV-2 immunoglobulin (Ig) G nucleocapsid antibody in summer 2020 were asked to complete an electronic survey focusing on their physical and mental health in Winter 2020–21. This survey was comprised of the Short Form 12v2, Physical Component Summary (PCS), Mental Component Summary (MCS), and Generalised Anxiety Disorder 7-item (GAD-7) questionnaires.7604/9781 (77.7%) HCWs were antibody tested, of which 1082 completed the full survey. Antibody testing was conducted between 17/06/20–30/07/20, during which time our seroprevalence rate was 28% (299/1082). Of those self-reporting COVID-19 symptoms, 51% (201/395) were antibody positive. Antibody-positive participants had lower PCS scores (p = 0.016), indicating poorer physical health. Lower PCS scores were also found in those deemed high risk for COVID-19 by their GP (p = 0.001), and those aged >44 years (p = 0.009). Antibody-negative participants had lower MCS scores (p = 0.044), indicating poorer mental health. Those who self-reported COVID-19 symptoms had lower PCS scores (p=<0.001) than those with no symptoms. Lower MCS scores were found in women (p = 0.001), Caucasians (p = 0.018), non-clinicians (p = 0.001), and those aged <44 years (p = 0.009). Significantly higher GAD-7 anxiety scores were evident in staff aged <44 years (p = 0.023), and those with self-reported COVID symptoms (p = 0.031). Doctors had lower GAD-7 anxiety scores (p = 0.009).Self-reported symptoms did not correlate with seroprevalence; data surrounding this can be useful for future workforce planning. Interventions are needed to reduce the mental and physical burden of the pandemic on HCWs. Further work is needed to identify which particular HCWs may require further support, to ensure well-being and effective patient care.Clinicaltrials.gov number -NCT04527432.Sponsor Protocol number - 2020COV112,  •Large study examining COVID-19 seroprevalence in healthcare workers (HCWs).•Links seroprevalence to physical and mental health-related quality of life scores.•Responses capture the impact from the first COVID-19 wave in the United Kingdom.•Set in an ethnically diverse National Health Service (NHS) Trust including all staff groups. The COVSeveral research studies and meta-analyses highlight the risks of depression, anxiety and insomnia in HCWs during the COVID-19 pandemic, but less is known about longer-term physical and mental health implications . There aThe COVID-19 Health Professional Impact Study (CHIP) study is a single centre cohort study, evaluating the impact of COVID-19 on the physical and mental health of a National Health Service (NHS) hospital workforce and the association between these and the presence of SARS-CoV-2 nucleocapsid antibodies, and the demographics and job types related to this.1.1There were two main aims of this study. Firstly, to determine the prevalence of SARS-CoV-2 antibodies in workers between 17th of June to July 30, 2020 (first wave of the pandemic), and how this compares to symptomatic self-reported COVID-19 infection. Secondly, to identify whether physical and mental health, including anxiety levels of HCWs relate to SARS-CoV-2 nucleocapsid antibody status, sex, age, ethnicity, job role , high risk COVID-19 status , and having symptoms of COVID-19.22.1This study was conducted at The Royal Wolverhampton NHS Trust (RWT), West Midlands, UK. In March 2020, RWT employed 9871 staff covering over 350 roles across three hospitals, including an 850 bedded secondary and tertiary care centre . Wolverh2.2All HCWs which included both clinical and non-clinical staff (support staff and estates teams included) were invited to participate in SARS-CoV-2 nucleocapsid antibody testing between 17th of June to July 30, 2020 . ResultsThis health survey component of the study ran from December 16, 2020 to March 15, 2021 . We inviFor data management and linkage, unique identifiers in the form of a mobile phone number or employee number were used to allow us to link SARS-CoV-2 nucleocapsid antibody status anonymously. Staff completed a web-based survey (appendix A). This included information on demographics, including age, sex, ethnicity, high risk COVID-19 status , occupation and whether this role was patient-facing or not, occurrence and dates of experiencing symptomatic COVID-19 infection as defined by the World Health Organisation , as COVIParticipants completed the Short Form 12v2 (SF12v2)  and the Data were collected in an anonymised manner and stored securely using SurveyMonkey (Survey Monkey Enterprise), a secure survey tool compliant with the Health Insurance Portability and Accountability Act (HIPAA). .2.3t-test to examine whether there were significant differences in the PCS, MCS and GAD-7 scores between those with versus without self-reported COVID-19 infection and COVID-19 antibody-positive participants versus negative participants. The third phase explored any significant differences in the PCS, MCS and GAD scores by each of the above-mentioned demographic classifications using independent samples t-test; from which we also report the effect size as Cohen's D measure. The fourth phase examined whether PCS, MCS, and GAD-7 scores were significantly influenced by key demographics and classifications in a stepwise multiple regression model is used to examine this. We used dummy coding for the categorical independent variables that included Gender , Ethnicity (Caucasian), Role (Doctor), COVID-19 Symptom (Self-Reported), Antibody Test (Positive), Patient Facing Role. All analyses were carried out in STATA® software version 16.1  number 287432 (appendix B). The study protocol (appendix C) is registered with 10.13039/501100000272National Institute for Health Research (NIHR) Clinical Research Network West Midlands. The Abbott SARS-CoV-2 IgG anti-nucleocapsid antibody test kits were provided free of charge. The sponsor, funder and Abbot laboratories had no role in study design, analysis or reporting of this study.The trial sponsors were RWT and the 3In March 2020, there were 9781 employees at RWT of which 7604 (77.7%) were tested for antibodies and were eligible for inclusion . 1504 HCBaseline characteristics of all respondents are shown in 1082 (89.7%) survey respondents were linked to their SARS-CoV-2 nucleocapsid antibody status. 395 (36.5%) of these had self-reported COVID-19 symptoms, of which 51% were, in fact antibody positive. The overall SARS-CoV-2 nucleocapsid antibody seroprevalence rate was 27.6% as of July 30, 2020. Out of participants who were seropositive, 67% had self-reported COVID-19 infection at or before the time of the survey .Table 2Sp = 0.031). The Cohen's D estimate showed medium effect for the significant difference in the GAD-7 scores.Of the HRQoL scores in p = 0.016); those with no symptoms of COVID-19 infection compared to those with symptoms (p = 0.009); those 44 years or younger (p = 0.009); and those not in the high-risk group for COVID-19 (p = 0.001) (924 (85%) participants completed the survey fully and could be linked to antibody data . Regress= 0.001) , Table 4p = 0.044), female staff (p = 0.001), people 44 years and below (p = 0.009), Caucasians (p = 0.018) and non-Doctors (p = 0.001) (p = 0.001), people 44 years or below (p = 0.023) and non-doctors (p = 0.009) (MCS scores were significantly lower in those who had negative antibody status (= 0.001) . GAD-7 w= 0.009) , Table 44Whilst the risks of COVID-19 infection in HCWs have been widely described, the CHIP study is one of the first, and largest, in linking symptomatic infection, antibody positivity and mental and physical HRQoL scores .4.1Individuals who were antibody positive or symptomatic had poorer self-reported physical health; this is consistent with data showing that those with poorer physical health are more at risk of contracting COVID-19  but may Compared to current normative data, the mental health of the HCWs in our cohort was worse . Our stuIn our study, those who were seropositive had significantly better mental health compared to those who were seronegative, which supports the findings of other investigations that have demonstrated a reduction in anxiety levels once an individual has contracted and subsequently recovered from the virus . IntriguThis current study also reinforces that females experienced worse mental health during the COVID-19 pandemic and similar results have been found in other studies , with wo4.2Having an awareness of seroprevalence can be useful from a workforce planning perspective and in reviewing infection control measures. Seroprevalence in other studies in HCWs greatly vary; the majority being between 11% and 33% and the highest rates being in New York and London . In compDespite the higher seroprevalence in HCWs than the general population, there is a need to confer long lasting immunity through vaccinations rather than relying on natural infection . Seropre4.3As the on-going pressures of COVID-19 within our communities become evident, understanding the mental and physical health impact of the pandemic will become increasingly important. The UK already has a lower doctor to population ratio and lower hospital bed per capita rate than many other European nations . FurtherIn terms of future work following vaccination, this study is being extended to capture further longitudinal data and to monitor the association between antibody positivity, COVID-19 infection rate and physical and mental HRQoL. During this study period, COVID-19 vaccination had only just begun, and antibodies were only tested prior to vaccination. In our extended study we will aim to address the impact of vaccination on seropositivity, mental and physical health scores and also the impact of physical and mental health on the antibody response to vaccination .4.4We aimed to recruit all staff as widely as possible by providing them with an individual personal SMS message. This was to capture a key but under-represented population in research, which is relatively deprived and ethnically diverse . HoweverThere was an inability to link participants’ wider electronic healthcare records to gain further demographic information. Some staff did not have their antibody results linked to their employee identification or mobile phone number. Staff without access to mobile smartphones would also have limited access, however, all staff are provided with IT and internet access on site. Further, the COVID-19 self-reported cohort is likely to include a number of cases related to other medical problems which were self-labelled as COVID-19. Although this is a limitation, the psychological and physical health impact of work loss, fear of case transmission, isolation and other factors would apply irrespective of case confirmation.Furthermore, we did not have baseline assessment of the HRQoL measures collected when the antibodies were assessed, making it difficult to fully ascertain if the increase or decrease of scores were related to COVID-19 infection. There are no large scale HRQoL scores available specifically for HCWs and the SF12v2 is norm referenced against the British population. In terms of mental health impact from COVID-19, there were many other factors that could be impacting this. This includes the negative effects of lockdown, social isolation and a personal history of mental health problems.We acknowledge that being vaccinated or not may constitute in assurance and relief from infection anxiety, but not depression. This was considered at the time of the writing of this manuscript, however, at the first phase of the study vaccination was not available. The emergence of new variants requires further research to plan new approaches.5The COVID-19 health professional impact study (CHIP) study examines in detail the impact the COVID-19 pandemic had on various subgroups of front-line healthcare workers (HCWs) in terms of mental and physical health. Future work will focus on subsequent COVID-19 waves and the vaccination stage.Our results are consistent with other studies revealing a significant impact of COVID-19 on the mental health of HCWs and association with inflammatory changes related to mental health conditions. Our findings further add to this by providing additional unique information on physical health and seroprevalence.Further research on how to combat the impact of COVID-19 is required, and we add to calls from other researchers in this area. Interventions are needed to address the long-term effects COVID-19 has had on the HCWs, who have and will continue to play a crucial role in fighting the pandemic.SS, NM and OS were involved in writing the study and manuscript revisions.AM did the statistical analysis of the study and manuscript revisions.HM, PB, AW and MD were involved in manuscript revisions.HSC, CF, AR were involved in the running of the study and in manuscript revisions.JB involved in protocol development, the running of the study, writing the study and in manuscript revisions.SP and JC involved in the concept of the study, protocol development, running of the study and in manuscript revisions.This work was supported by National Institute of Health and Care Research (sponsor number 2020COV112). Abbott Laboratories provided the SARS-CoV-2 Immunoglobin (Ig) test kits used in this study.Abbott Laboratories UK provided the SARS-CoV-2 Ig test kits but had no role in the design or manuscript development of the study.None of the authors have relevant conflicts of interest to declare.


Background: Life expectancy for patients with malignant tumors has been significantly improved since the presence of the programmed cell death protein-1/programmed cell death protein ligand-1 (PD-1/PD-L1) inhibitors in 2014, but they impose heavy financial burdens for patients, the healthcare system and the nations. The objective of this study was to determine the survival benefits, toxicities, and monetary of programmed cell death protein-1/programmed cell death protein ligand-1 inhibitors and quantify their values.Methods: Randomized controlled trials (RCTs) of PD-1/PD-L1 inhibitors for malignant tumors were identified and clinical benefits were quantified by American Society of Clinical Oncology Value Framework (ASCO-VF) and European Society for Medical Oncology Magnitude of Clinical Benefit Scale (ESMO-MCBS). The drug price in Micromedex REDBOOK was used to estimate monthly incremental drug costs (IDCs) and the correlation between clinical benefits and incremental drug costs of experimental and control groups in each randomized controlled trial, and the agreement between two frameworks were calculated.Results: Up to December 2022, 52 randomized controlled trials were included in the quantitative synthesis. All the randomized controlled trials were evaluated by American society of clinical oncology value framework, and 26 (50%) met the American society of clinical oncology value framework “clinical meaningful value.” 49 of 52 randomized controlled trials were graded by European society for medical oncology magnitude of clinical benefit scale, and 30 (61.2%) randomized controlled trials achieved European Society for Medical Oncology criteria of meaningful value. p-values of Spearman correlation analyses between monthly incremental drug costs and American society of clinical oncology value framework/European society for medical oncology magnitude of clinical benefit scale scores were 0.9695 and 0.3013, respectively. In addition, agreement between two framework thresholds was fair .Conclusion: This study suggests that there might be no correlation between the cost and clinical benefit of programmed cell death protein-1/programmed cell death protein ligand-1 inhibitors in malignancy, and the same results were observed in subgroups stratified by drug or indication. The results should be a wake-up call for oncologists, pharmaceutical enterprises and policymakers, and meanwhile advocate the refining of American Society of Clinical Oncology and European Society for Medical Oncology frameworks. Survival benefits of patients with a malignant tumor have been improved significantly over the years, partially attributed to the employment of novel anti-cancer therapies. Recent success in immunotherapy propels cancer treatment to an exciting new era after traditional chemotherapy and targeted therapy . To dateHowever, it is always hard to objectively quantify therapy value and clinical benefit. It is commendable that the American Society of Clinical Oncology Value Framework (ASCO-VF)  and the Thus, this study attempted to employ the ASCO-VF and the ESMO-MCBS to describe the clinical benefit of all approved PD-1/PD-L1 inhibitors for treating malignant tumors, and calculated the unit time cost of each agent, so as to explore a correlation between clinical benefit and price of drugs, likewise the correlation analyses in the subgroups of different agents or indications. Furthermore, consistency evaluation of two value frameworks was also computed.clinicaltrials.gov website were also incorporated. Abstracts and methods of each trial were reviewed to identify the eligible cohort of trials according to inclusive criteria that RCTs could be analyzed with ASCO-VF or ESMO-MCBS, and the clinical benefit of the experimental groups should be preferred over the control groups. Analyses of patient-reported outcomes only assessing the QOL of the corresponding RCTs were also included. The following research were not taken into account: the secondary, subset, or systematic reviews; phase I, II or IV trials or animal studies; trials focused on other objectives including pharmacokinetics, drug dosing schedules, iconography, biomarkers, modeling, etc; non-trial-based papers like trial introduction; and trials that written by non-English articles. The study was conducted independently by two authors (YH and SL), and discrepancies were resolved by consensus in the presence of a third investigator (XW).PubMed was searched from the inception of a database to December 2022 to identify all the phase III randomized controlled trials (RCTs) in treatment with malignant tumors involving approved PD-1/PD-L1 inhibitors , by the terms of drug names and clinical trials . Phase III RCTs registered on the Gains in a survival endpoint and adjustments by toxicity and QOL in scores or grades were quantified by ASCO-VF (Version 2), or ESMO-MCBS (Version 1.1), or both if data allowed. The clinical benefit score of ASCO-VF is based on the point estimate of the hazard ratio (HR) of a couple of clinical endpoints covering overall survival (OS), progression-free survival (PFS), and response rates (RR), which is subtracted from 1 and the result is multiplied by 100 to derive the preliminary score. For toxicity assessment, both the number of the occurred case and the frequency  of all grades’ adverse events are correspondingly assigned “points”, which are applied to formulaically figure up the increment of the experimental group against the control group to derive an adjustment of the score . For QOL, ASCO-VF allows an award of 10 points if a statistically significant improvement in QOL is reported but no deduction due to detrimental QOL. Besides, ASCO-VF includes bonus points for a “tail of the curve effect” (16–20 points), palliation of symptoms (10 points), and treatment-free intervals . The final ASCO-VF scores are the sum of above items (possible range −20–180) . ASCO-VFIn the ESMO-MCBS grading system, the lower limits of the 95% CI of the HR of survival outcomes are used to determine a particular grade in a pre-specified manner, which is downgraded if pre-specified toxic effects are explicitly outlined in the experimental group with specifically, statistically significant incremental rates like “Toxic death >2%,” “Cardiovascular ischemia >2%,” “Grade 3 neurotoxicity >10%” and so on. For QOL assessment, upgrading or downgrading are allowed base on the improvement or deterioration of QOL. Ultimately, ESMO-MCBS grades are ranked from 1 to 5 for the advanced disease setting, and C, B, or A for the curative setting. ESMO-MCBS defines “meaningful clinical benefit” as a grade of 4, 5 or B, A .2 and creatinine clearance of 100 mL/min  for drugs from the RedBook . Monthly costs were calculated over an average of 28 days based on the dosage schedule in all eligible trials for a patient weighing 70 kg with a body surface area of 1.86 m0 mL/min . Ultimatr) was calculated to assess the association between non-normally distributed data or ordinal data, such as costs and scores, which were showed by scatterplots or boxplots. Mann-Whitney U test was performed to describe the correlation between cost data and clinical benefit thresholds, shown as boxplots. Agreement between ASCO-VF and ESMO-MCBS in clinical benefits of RCTs was calculated via Cohen κ statistics, by which the result was between 0 and 1   . No quanp-values of less than 0.05 were considered statistically significant.All statistical analyses were conducted in R (version 4.1.0) using ggplot2 (version 3.2.0) for plots. A total of 101 RCTs were initially identified. After excluding trials that failing to meet inclusion criteria, 65 papers of 52 phase III RCTs were analyzed containing six PD-1/PD-L1 inhibitors: 17 RCTs for pembrolizumab, 14 RCTs for nivolumab, 14 RCTs for atezolizumab, 4 RCTs for durvalumab, 2 RCTs for avelumab, 1 RCTs for cemiplimab . The RCTOf 52 RCTs, 4 were the curative setting, and the others were the advanced setting. All-inclusive RCTs were eligible for assessment by the ASCO-VF, and among which 49 RCTs were also eligible for ESMO-MCBS assessment.ASCO-VF scores ranged from 0.40 to 86.71 . The scoFor the assessment of ESMO-MCBS, among 49 RCTs, 19 trials felled below the “meaningful benefit” score, 30 were above . For theρ = 0.0054, p = 0.9695, ρ = −0.0396, p = 0.3946), and curative treatments   of PD-1/PD-L1 inhibitors and the ASCO-VF score were not statistically significant correlated in all trials (Spearman’s = 0.184) .ρ = −0.0788, p = 0.3013) (p > 0.05)  . Stratif > 0.05) . Correlap = 0.8444], while the opposite result was observed when used ESMO-MCBS framework   vs. $13392 (IQR 9391 to 26681);  0.9014] . Neitherκ statistic was calculated as 0.417 (p = 0.00354), which suggested a moderate agreement between ASCO-VF and ESMO-MCBS thresholds. In subgroup analyses, within the palliative subset, the κ score  was similar to that in the total cohort, whereas in curative setting of trials, the κ score was weaker than the total cohort .When comparing the RCTs scores using the framework-specified thresholds, Cohen Cancer drug innovation has been accelerating since entering the 21st century. The number of novel cancer drugs approved in 2005–2015 was over 8 times more than that approved in 1975–1985 (66 vs. 8), and the average annual growth rate of total cancer drug expenditure was 7.6%, 3.6 times more than the average annual growth rate of nominal United States Gross Domestic Product . In the Previously, two prior studies showed no significant association between clinical benefit and the price of new FDA-approved anti-cancer drugs with initial indications in the United States from 2000 to 2017, using both ASCO-VF and ESMO-MCBS . The resTwo value frameworks were applied in this study, and a moderate agreement was found between them. There is a controversy exist in the agreement of these two frameworks , which aFrom the perspective of society, growing expenditures on anticancer drugs can potentially occupy the investment of other life–saving medicine, and contribute to the unbalanced allocation of medical resources. Virtually, many drugs like anti-cardiovascular diseases drugs are available as generics or “me-too” that are defined a new pharmaceutical compound with a known pharmaceutical class of treatment, and increasing competition consequently led to diminishing overall costs in these pharmaceutical companies, while most the anticancer agents are the first-in-class agents. During 1970–2000, the life expectancy of Americans increased on average by 6 years; only 6 months were attributed to antineoplastic therapies, while over 4 years were attributed to cardiovascular disease . A horreThis study has several limitations. Firstly, we only evaluated available trials published to assess the ASCO-VF and the ESMO-MCBS scores to date. Within a trial, outcomes of long-term follow-up and the further pooled estimate of efficacy result would evolve with time, which lead to the dynamics of clinical benefit scores of drugs . SimilarThis research indicated that on account of ASCO-VF and ESMO-MCBS frameworks, no correlation between the costs and clinical benefits of PD-1/PD-L1 inhibitors was present in treating malignant tumors, and the same results were observed in subgroups stratified by drugs or indications. In addition, the agreement between two framework thresholds was moderate. The result suggests that a comprehensive cost-benefit assessment of novel cancer drugs should guide oncological drug approval in public healthcare organizations, and methods to control and limit drug cost should be coordinated among healthcare providers, pharmaceutical companies, and policymakers. Meanwhile, the refining of ASCO and ESMO frameworks might be addressed to facilitate the standard assessment of clinical benefit of anti-cancer drugs.


FLT3 gene, termed FLT3-ITDs, are useful molecular markers in acute myeloid leukemia (AML) for patient risk stratification and follow-up. FLT3-ITDs are increasingly screened through high-throughput sequencing (HTS) raising the need for robust and efficient algorithms. We developed a new algorithm, which performs no alignment and uses little resources, to identify and quantify FLT3-ITDs in HTS data.Internal tandem duplications in the k-mers from reads covering FLT3 exons 14 and 15. We show that those k-mers bring enough information to accurately detect, determine the length and quantify FLT3-ITD duplications. We compare the performances of FiLT3r to state-of-the-art alternatives and to fragment analysis, the gold standard method, on a cohort of 185 AML patients sequenced with capture-based HTS. On this dataset FiLT3r is more precise  than the other software evaluated. We also assess the software on public RNA-Seq data, which confirms the previous results and shows that FiLT3r requires little resources compared to other software.Our algorithm (FiLT3r) focuses on the https://gitlab.univ-lille.fr/filt3r/filt3r. The repository also contains a Snakefile to reproduce our experiments. We show that FiLT3r detects FLT3-ITDs better than other software while using less memory and time.FiLT3r is a free software available at The online version contains supplementary material available at 10.1186/s12859-022-04983-6. FLT3 gene (FLT3-ITD), which occurs in 20-30% of the cases [FLT3-ITD are in-frame duplications of highly variable size, ranging from 3 to more than 400 nucleotides, mostly located within the receptor’s autoinhibitory juxatamembrane domain. This mutation represents a strong prognostic biomarker since patients with FLT3-ITD are at higher risk of relapse and have decreased event-free and overall survival [FLT3-ITD has a major therapeutic impact, such as indication of allogeneic hematopoietic stem cell transplantation  in first complete remission [FLT3-ITD is also a therapeutic target with the emergence of FLT3 inhibitors, used in combination with chemotherapy during induction and consolidation courses [Monitoring the disease progression in blood cancers requires the identification of pathology-specific molecular markers. In acute myeloid leukemia (AML), one of the strongest risk factor used for risk stratification is the presence of an internal tandem duplication in the he cases –4. FLT3-survival . The impsurvival . Presencemission –8. FLT3- courses , 10 and  courses .FLT3-ITD were performed with fragment analysis [FLT3-ITD. Thus, many methods were specifically developed to detect FLT3-ITD in high-throughput sequencing data [Historically, according to European LeukemiaNet (ELN) guidelines, the identification and quantification of analysis . DNA fraanalysis , 13. Oneanalysis , 15,  is very short. With short values of k, there may exists several occurrences of the same k-mer in the reference sequence  with the k-mers from the reference sequence is above a given threshold to prevent spending time on reads not coming from the gene of interest. For each of these reads we will now focus on the k-mers occurrences in the reference sequence. We call trace ofRinT, denoted by k-mers positions in T from a read R. Then k-mer T  the maxR, this will create a break in the trace. Note that the converse is not true: many other events can also create breaks. Actually, any difference between the read and the reference sequence will lead to a break. Also, in some rare cases, the duplication will not create a break. However, the probability of this phenomenon decreases exponentially with k.Whenever a duplication occurs in a read g is the size of the break. In such a case, a duplication has been detected . Thus, the computed duplication size won’t change compared to the situation where no such substitution occurs. Thus, our heuristic is robust to such events.The length of the duplication can be deduced from the mentclass2pt{minimWe are finally able to identify duplications within a read in linear time. In order to limit the false positives, we introduce several strategies to mitigate this risk.k-mers are short sequences that can occur by chance because of sequencing errors, or other events. We must not detect a duplication whenever a (few) short k-mers occur in the wrong place.False positive detection of duplication can be encountered because k-mers may occur at several locations in the reference sequence. Of course only one (at most) is correct, thus only one is kept. Assuming we want to disambiguate ie.First, if ie. the absolute values of the subtractions) with the neighbouring positions in i is just before or just after a break.Otherwise, among the possible positions in In the read trace, some Substitutions are the most frequent mutations and sequencing errors . Therefk-mer starting at position k-mer doesn’t exist in T, as position k-mer. If one of the corrected k-mer occurs at a position p such that k-mer this way, and we move on to the next k-mer. In case the correction did not work, the k-mers in the break are not corrected.The correction is carried out as follows: let a break k-mers. Thus, if we have a candidate break Before reporting a duplication event, we make sure this event is robust by checking that ie.Moreover, if two breaks Identifying a duplication is not sufficient, as its abundance is also a prognosis factor. Rather than a raw abundance, a relative abundance is much more meaningful. We express the relative abundance as a VAF (Variant Allele Frequency) or an AR . All the duplications sharing the same characteristics (same length and same starting and ending positions) are pooled together.Briefly speaking, the abundance is computed from the number of reads in which a given duplication is found and the number of reads corresponding to the wildtype sequence. The read counts is further corrected to take into account the duplications that cannot be observed when they occur at one end of the reads.ie. the number of reads that cover the position of the duplication in the reference sequence and in the duplicated version. To do so, we use the disambiguated traces. The traces provide the positions that were covered in the reference sequence, we can thus count the coverage at each position in the reference. More formally, when j in the reference sequence. This corresponds to the total coverage, which thus includes the coverage of the wildtype as well as the coverage of all the duplications or indels detected. We deduce the wildtype coverage by removing all the coverages from the duplications/indels that have been detected.More precisely, to determine the VAF or the AR we need to know, at the position of the duplication, both the coverage of the wildtype version and of the duplicated version, b, we will need to have R is the size of the reads. The counts are corrected to take this observation into account, and by assuming that any indel has a uniform probability of occurrence within the read. Let q be the raw quantification, ie. the number of reads in which the duplication was identified, the corrected quantification is However, with our approach we cannot detect duplications that would occur at the start or at the end of a read. As explained previously, for a duplication to be detected, we need to have k-mers coming from the reference sequence. However, in most cases most of the reads would not come from the region of interest. Thus, the most time-consuming step in our algorithm is to determine whether the read is coming from that region. To lower this time consumption, we use a heuristic: k-mers from the region of interest are stored in a Bloom filter [As specified previously, we only search duplications in reads having enough m filter .k-mers come from the region of interest in the read. If this value is above a threshold then the read goes to the following stages of the algorithm, otherwise it is discarded. By default, this threshold is set at 30 %.The first step is therefore to query the Bloom filter to determine how many FLT3 gene, is quite short the Bloom filter can be very small and will likely be stored in the CPU cache, which will allow very quick accesses.Bloom filters do not yield any false negative, therefore we have the guarantee that this heuristic will not prevent us from identifying a read that actually comes from the region of interest. As the region of interest, namely exons 14 to 15 from the gitlab.univ-lille.fr/filt3r/filt3r.FiLT3r is implemented in C++ using the GATB library  and is fFLT3 and can more generally detect variations in raw reads. FLT3-ITD-ext relies on a general-purpose aligner .We compared FiLT3r with state-of-the-art approaches. We made a preliminary assessment on our cohort with several other software detecting FLT3-ITD-ext, such as pindel , ITDSeekBWA-MEM, ) to aligk-mer size of 12k-mer size of 31 (as advised by km authors) and with a threshold for duplication detections of .01 , Jellyfish parameters were identical to the ones used in km publication (-C -L 2 -Q+ -s 1000000). FLT3-ITD-ext was launched with default parameters. For FLT3-ITD-ext, the deletions were not considered as it would have lead to too many false positives. Within a sample, all the duplications of the same length are pooled together to take into account the fact that the allelic ratio method only differentiates duplications on their lengths. The experiments can be reproduced with a Snakefile available on the Git repository of the software.FiLT3r was launched with a All the experiments were performed on a server with 24 Intel®Xeon®CPU E5-2420 and 193 GB of RAM. The data was stored on a NAS connected to the server through NFS. The programs were run on a single thread. User times and peak memory consumptions were measured.de novo or secondary AML at the haematology laboratory of the University Hospital of Lille. This population was composed of 114 FLT3-ITD positive patients and 71 FLT3-ITD negative patients, according to the gold-standard fragment analysis [We assessed the three software packages on high-throughput sequencing data of 185 patients aged from 18 to 60 years old, diagnosed with analysis . The stuFLT3 were sequenced in all patients using the following methodology: the library was prepared by a capture method, targeting 62 genes with SureSelect-QXT (AGILENT®) according to the manufacturer’s protocol. Samples were sequenced with NextSeq Illumina® with an average read length ranging from 68nt to 150nt (median: 101nt).To assess how the three software behaves on datasets with very different characteristics, we used public RNA-seq data. RNA-seq is gaining importance in clinical settings and RNA-seq data differs widely from capture sequencing in terms of quantity of data, variation of expression and random starting positions of the reads. We launched the three software on data from CCLE (Cancel Cell Line Encyclopedia) . We randFinally, to check that our results holds on controlled data, we simulated ITDs at different ratios with different qualities.The software results on HTS data were compared to DNA fragment analysis, the gold-standard method. Any duplication found by a software, at whatever abundance, and identified with the reference method was considered as a true positive. Any duplication found by a software with an allelic ratio (AR) above .01 and that was not identified by the reference method was for now considered as a false positive. After analysing the results in details, we will change this definition of false positivity as HTS appear to be more sensitive to detect lowly expressed ITDs.Raw results are shown in Table However, some 7/17) of the false positives were shared with either km or FLT3-ITD-ext. We repeated a fragment analysis on the two samples where the false positives were the most abundant, to assess whether they were real false positives. We eventually obtained a sequence by Sanger for both  but with low concentrations. All the false negatives but one has a quantification It is noteworthy that all the false positives were identified in positive samples (apart from one of the Sanger-confirmed duplication) and at lower ratios than the duplication identified by the reference method. Thus, we consider that our original definition of Beyond the correct detection of ITDs, it is important to also correctly assess the quantification of those duplications as they are meaningful for the prognosis of the patients. Figure k-mers, with Jellyfish, which is a required step for km.Regarding time and space consumption, FiLT3r showed the best performances  is 6 times quicker than Jellyfish’s and 9 times quicker than FLT3-ITD-Ext’s. In some cases FLT3-ITD-Ext can be very time-consuming, taking several hours, while FiLT3r never took more than 10 minutes. The progression margin for FiLT3r is quite low as gunzip on the same files took about a third of the time took by FiLT3r , FLT3-ITD-ext had to be killed before the results were output as it took too much time (more than 24 h) or too much memory (more than 100 GB).As expected none of the software detected a FLT3-ITD were detected by the three software. At the same 0.01 threshold as for the capture sequencing, FiLT3r detected 3 ITDs that were also detected by km, although one of them was only detected by km with -p 0.001 not with -p 0.01. FLT3-ITD-Ext detected the remaining two.On the 76 haematological samples, some km additionally detected a 60nt ITD in SRR8657348 that was not detected neither by FiLT3r nor FLT3-ITD-Ext. However this 60nt duplication seemed to be an artifact from a 30nt duplication that was actually detected by the three software. km seemed to detect a duplication of the duplication, however we found no read with evidence of such an event.FLT3-ITD-ext detected three ITDs above the .01 threshold, two of which were not detected by FiLT3r or km: a 108nt ITD in SRR8615750, and a 105nt ITD in SRR8615696. The third ITD (a 78nt duplication in SRR8615696) was also detected by FiLT3r but at a much lower AR (.0007 instead of .02). In this sample we found evidence of only two reads containing the ITD breakpoint, casting doubt on the .02 AR given by FLT3-ITD-Ext. Moreover, km did not detect this ITD but was launched with options -p 0.01 or -p 0.001 and thus could not detect a very lowly expressed ITD. Regarding the 108nt ITD only found by FLT3-ITD-Ext, we found evidence of such a breakpoint in only one read. Concerning the 105nt ITD, the sequence reported by FLT3-ITD-Ext does not align in full-length on the reference, only the first 70nt do align. The last 35nt did not align on NCBI Blast non-redundant nucleotide sequences either. See Additional file Overall, FiLT3r completed each task in less than 3 hours with less than 32 MB or RAM (median: 344 s and 26 MB). km (including Jellyfish step) took at most 9.5 hours and 37 GB of RAM for one sample (median: 962 s and 2.5 GB). FLT3-ITD-ext median time was 4400s with a median memory usage of 799 MB of RAM.in silico using art with the MiSeq v3 profile of errors [We simulated FLT3-ITDs using itdsim which is part of ITDseek . Then thf errors . The detWe introduced FiLT3r, a time and memory efficient algorithm implemented in an open-source C++ software, which showed very good detection and quantification performances compared to state-of-the-art software. On our capture dataset, FiLT3r had a perfect recall, which was not the case of the other software. Even the reference fragment analysis method, exhibited two false negatives, as shown by further Sanger sequencing of the samples. Similarly to the other software, FiLT3r had a perfect precision. FiLT3r performances were better than that of FLT3-ITD-ext, the best software identified so far by an independent benchmark, which is alignment-based. Moreover FLT3-ITD-ext could not run on several RNA-seq dataset due to large resource requirements (both time and memory). In two cases on RNA-seq dataset, FLT3-ITD-ext reported quantification that appeared largely overestimated.FLT3-ITD analysis, FiLT3r can also be used to detect duplications in any gene as soon as the reference sequence is known. We plan to apply our method to other genes in other contexts, as it has been shown that tandem duplications can be prognostic signatures in other cancers .Additional file 1. supp file 1: filt3r-supp.pdf.Additional file 2. supp file 2: results_sra.xlsx.Additional file 3. supp file 3: accessions.tsv.Additional file 4. supp file 4: results-different-k.xlsx.Additional file 5. supp file 5: results-small-k.xlsx.Additional file 6. supp file 6: results-different-perc.xlsx.Additional file 7. supp file 7: results-subsamples.xlsx.Additional file 8. supp file 8: genescan_SRR15006459.pdf.Additional file 9. supp file 9: genescan_SRR15006372.pdf.Additional file 10. supp file 10: accessions-CCLE.csv.Additional file 11. supp file 11: results_simulated_150bp.xlsx.Additional file 12. supp file 12: results_simulated_250bp_hq.xlsx.Additional file 13. supp file 13: results_simulated_250bp_lc.xlsx.Additional file 14. supp file 14: results_simulated_250bp_lq.xlsx.Additional file 15. supp file 15: results_simulated_250bp.xls.


Apis mellifera) are critical to maintaining ecological balance and are important pollinators. The oviposition behavior in honeybees is important and complex. Circular RNAs (circRNAs) are found to form circRNA-miRNA crosstalk and play important roles in reproduction processes. Here, dual luciferase reporter was used to confirm the crosstalk between ame_circ_2015 and ame_miR-14-3p. Functional experiments in vitro and in vivo were performed to investigate the biological functions of ame_circ_2015 in egg-laying of queens. The results showed that ame_circ_2015 directly target ame_miR-14-3p, and the expression of ame_circ_2015 was negatively correlated with ame_miR-14-3p expression. Overexpression results showed that ame_circ_2015 promoted the number of eggs laid and knockdown of ame_circ_2015 suppressed the number of eggs laid. It demonstrates that up-regulated ame_circ_2015 promotes the number of eggs laid by sponging ame_miR-14-3p. The study will provide information towards a better understanding of circRNA-miRNA crosstalk in egg-laying in honeybees.Honeybees ( Apis mellifera) are important pollinators and are critical to ecology balance [Honeybees  is a new type of non-coding RNAs ,4. CircRcificity ; in 2015cificity ; in 2017cificity . Our precificity . Howeverp < 0.05) in the ovary activation process [In this study, we focus on one circRNA, ame_circ_0002015  . The mixed sperm were collected from 1000 drones from three different queens’ families. After successfully laying eggs, each queen was introduced to its own colony. Each colony has the similar size. The number of eggs laid by each queen was checked and around 800 eggs per day were observed. All queens were included in the next siRNA injection experiment.The honeybee colonies used in the study were maintained by Institute of Apicultural Research, Chinese Academy of Agricultural Sciences , Beijing, China. More than 100 Each queen was injected with 1 μL siRNA solution  or 1 μL nonsense sequence. The procedure of injection was implemented according to Amdam’s methods . In specTotal RNA was extracted using Trizol reagent  according to the manufacturer’s instructions. The purity, concentration and integrity of RNA were assessed, and all qualified RNA was used in next experimental protocol.18 primer. Then, transcript quantification was performed  and oligo (dT)erformed . The RT-For first strand cDNA synthesis of miRNA, reverse transcription reaction was performed using miRcute Plus miRNA First-strand cDNA Kit . Then, transcript quantification was performed using miRcute Plus miRNA qPCR Kit (SYBR Green). An amount of 5 μL of cDNA  was included in a total volume of 20 μL. U6 served as the internal reference gene.−ΔΔCt method.By standard curve calculation, PCR efficiency of each gene was estimated. Ct values were transformed to quantities and relative gene expression was calculated using the 2Drosophila S2 cells were used in the study. Cultured with 24 h, miR-14-3p agomir/antagomir was co-transfected with either psiCHECK2-circ_2015, psiCHECK2- circ_2015-Mut, or an blank vector as control in the cells using the method as described by Tiscornia et al. [To test whether miR-14-3p actually targets circ_2015, a luciferase assay was performed using a dual-specific luciferase assay kit . Two luciferase reporter plasmids were designated, named as psiCHECK2-circ_2015 and psiCHECK2-circ_2015-Mut. For psiCHECK2-circ_2015, we sub-cloned the complete sequence of circ_2015 (1142-bp) including the predicted miR-14-3p recognition site. For psiCHECK2- circ_2015-Mut, the predicted miR-14-3p recognition site was changed to mutations. For cell culture, a et al. . Twenty-To overexpress circ_2015, an overexpressed vector was constructed. The complete sequence of circ_2015 was cloned into a pcDNA3.1 vector and amplified using PCRMasterMix  . To inhiA one-way ANOVA and a t-test were conducted to analyze the RNA expression in different groups. Data were analyzed by software SPSS 22.0 .p < 0.01, p < 0.05, The results of luciferase reporter assay confirmed the interaction between circ_2015 and miR-14-3p. It showed that the luciferase activity significantly decreased in the group of miR-14-3p agomir co-transfected with psiCHECK2-circ_2015 compared to the group of miR-14-3p agomir co-transfected with psiCHECK2-circ_2015 Mut (p < 0.01). The results showed that the inhibition caused by siRNA was effective (p < 0.05), and was increased around 3.1 times at 48 h  and decreased by 72% at 48 h (p < 0.01), respectively (p < 0.05) and increased by 300% at 48 h (p < 0.01), respectively (p < 0.01) and increased around 4.4 times at 48 h (p < 0.01), respectively (p < 0.01) and decreased by 57% at 48 h (p < 0.01), respectively . The expression of circ_2015 decreased by 56% the siRNA group compared with that of the siRNA NC group , while circ_2015 expression from the siRNA group decreased by 56% compared with that of the siRNA NC group   b. The eg < 0.05) b. The rep < 0.05), but not significantly between the 5.0 μg and the 7.5 μg group. The number of eggs laid did not increase between the 7.5 μg group and the 10.0 μg group.To further confirm the circ_2015 titer-dependent effect on the number of eggs laid, four groups with different amount of synthetic reagent were implemented c. The reHoneybees are an indispensable species for maintaining the sustainable development of the ecological environment. One of the important traits of honeybee queens is egg-laying. Excellent queens with a high eggs-laid number can rapidly develop the colony size. This will make a good preparation for the further pollination and production. However, the genetic architecture of reproductive traits is complex. Additionally, the heritability is low. The eggs-laid number cannot be measured until queens’ sexual maturity and this makes early selection impossible. By using traditional selection methods, the genetic improvement would be slow. Fortunately, with the development of molecular biology, there are new methods to help improve complex traits, such as molecular marker-assisted selection and genome selection . Using tCircRNAs play an important role in various biological processes and regulate genes’ expression. With the development of molecular biology, more studies on circRNAs’ roles in reproduction processes were reported. For example, Song et al. found that the expression of circRNAs was related with estrogen(E2)/progesterone(P4) and affect the endometrium receptiveness during embryo implantation in goats . Zhang eTo determine whether the expression of circ_2015 was directly associated with the eggs-laid number of queens, we recorded the number of eggs laid per day by each queen and analyzed the association between the circ_2015 expression and the number of eggs laid by queens. The results indicate that the overexpression of circ_2015 promotes the number of egg-laid and the inhibition of circ_2015 repress the number of egg-laid. Thus, we deduce that there is a good correlation between the expression of circ_2015 and the number of eggs laid of queens. Next, we detected whether the circ_2015 titers was related with the number of eggs laid. The results showed that accompany with the increasing of circ_2015 titer, the number of eggs laid increased too. In our previous study, we also reported that circ_2015 was only found in queens’ ovaries instead of workers . The resdrosophila, zinc finger protein ush is dynamically expressed during embryonic processes. It was reported that the Ush transcriptional regulator played a critical role in embryogenesis in drosophila [Apis mellifera with Drosophila melanogaster is high, so each genes’ function may be similar too. Our previous study also found that zinc finger protein ush was up-regulated in ovarian activation process [The possible mechanism of circ_2015 affecting the number of egg-laid in queens was analyzed in two directions. Firstly, we analyzed the parental gene of circ_2015, which is zinc finger protein ush (LOC100577801). Multitype zinc-finger proteins of the Friend of GATA/U-shaped (Ush) regulated genes’ expression by their modulation of GATA factor activity. In osophila . The gen process . Additio process . TherefoThe second possible mechanism of circ_2015 affecting oviposition is circRNA-miRNA crosstalk. CircRNAs can competitively bind miRNAs through sequence-binding sites, thereby releasing miRNA’s inhibitory effect on its target gene, and finally regulating the target genes’ expression ,28. In hWith the development of molecular biology technology and the discovery of a large number of biological genetic markers, the genetic and breeding of honeybees has been further developed. In recent years, studies showed that a complex interaction network formed by coding and non-coding RNA affect the egg-laying of honeybees ,32,37,38In summary, we detected that circ_2015 sponged miR-14-3p, and the overexpression of circ_2015 enhanced the number of eggs laid of queens. These findings suggested that circ_2015, by sponging miR-14-3p, played an important role in regulating egg-laying of honeybee queens.


When the E gene Ct cutoff was 30.0, 25.0, 20.0, and 15.0, the sensitivity of the RAT was 71.9%, 80.3%, 93.0%, and 97.8%, respectively. The sensitivity of the RAT could be considered high in patients with a high viral load, and the RAT could be used as a screening tool in the ED.The rapid antigen test (RAT) has been adopted as a screening tool for SARS-CoV-2 infection in many emergency departments (EDs). We aimed to investigate the diagnostic value of the accuracy of the SARS-CoV-2 RAT as a screening tool in the ED. This retrospective observational study included patients who underwent both RAT and RT–PCR and visited the ED from 1 December 2021 to 15 March 2022. RAT and RT–PCR were performed by appropriately trained physicians. We performed detailed analyses using the E gene cyclic threshold (Ct) values of RT–PCR. Out of a total of 1875 patients, 348 (18.6%) had positive and 1527 (81.4%) had negative RT–PCR results. The overall sensitivity, specificity, positive predictive value, and negative predictive value of the RAT were 67.8%, 99.9%, 99.6%, and 93.2%, respectively. The E gene Ct value was significantly lower in the RAT-positive patients than in the RAT-negative patients (18.5 vs. 25.3,  The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was initiated in China in late December 2019, and coronavirus disease 2019 (COVID-19) has progressed as a global pandemic . The varThe early identification and isolation of COVID-19 patients to prevent the spread of SARS-CoV-2 infection is crucial, especially in the emergency department (ED) . PatientReal-time reverse transcription polymerase chain reaction (RT–PCR) is currently used as a diagnostic gold standard for the detection of SARS-CoV-2 infection ,10. NuclThe RAT has been adopted as a screening tool for SARS-CoV-2 infection in many EDs ,20,21,22This retrospective observational study was conducted in the ED of Hanyang University Hospital in Seoul, Korea. This study was approved by the Institutional Review Board (IRB) of Hanyang University Hospital (IRB No. HYUH 2022-03-037). The requirement for informed consent was waived because the data were entirely retrospectively collected.We included patients who visited the ED from 1 December 2021 to 15 March 2022. From December 2021, we introduced a protocol to perform RAT on almost all patients who visited the ED. The identification of viral RNA using RT–PCR is the gold standard for identifying SARS-CoV-2 infection. Patients who underwent RAT and concurrent COVID-19 PCR were included in the study. We performed RT–PCR along with RAT for ED patients with symptoms or signs related to COVID-19 or who needed hospitalization.RAT and RT–PCR swabs for COVID-19 were performed by appropriately trained physicians. RAT was performed using samples collected from nasopharyngeal swabs. RAT was performed with the STANDARD™ Q COVID-19 Ag Test . RAT is a rapid chromatographic immunoassay for the qualitative detection of SARS-CoV-2 nucleocapsid antigen. The COVID-19 antigen test cassette is coated with two lines: a control line and a test line. The control line was coated with mouse monoclonal anti-chicken IgY antibody, and the test line was coated with mouse monoclonal anti-COVID-19 antibody. If the SARS-CoV-2 antigen is present in the sample, the purple line is displayed on the test line. The control line is always displayed when the sample migration is successful. Visual interpretation of the results was performed between 15 and 30 min.RT–PCR was performed using samples collected from both nasopharyngeal and oropharyngeal swabs. Two RT–PCR assays were conducted as a reference standard test to confirm the presence of COVID-19. PowerCheck™ 2019-nCoV RT–PCR  was performed in hospital during weekdays. The AllplexTM 2019-nCoV Assay  was performed as an outsourced test at night and on weekends. The cyclic threshold (Ct) cutoff indicating COVID-19 positivity was ≤38 for the PowerCheck™ 2019-nCoV RT–PCR and ≤40 for the AllplexTM 2019-nCoV Assay. The Ct value of COVID-19 has been reported for the E, RdRP/S, and N genes; E and ORF1ab gene Ct values in Omicron patients. Therefore, the E gene Ct values in patients with COVID-19 have always been reported, regardless of whether they were Omicron patients. For this reason, we performed various detailed analyses using the E gene Ct values.t-test or Wilcoxon rank sum test was performed depending on the results of the normality test.Categorical variables are presented as numbers and percentages. Fisher’s exact test was performed when comparing the categorical variables of two groups. The normality test of continuous variables was performed using the Shapiro–Wilk test. Continuous variables with normality are presented as the mean and standard deviation, and variables with nonnormal distribution are presented as the median and 25th to 75th percentiles. Student’s p value was less than 0.05 on a two-sided test. All statistical analyses were conducted using R .Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated using 2 × 2 tables for the RAT and RT–PCR test results and are presented as % and 95% confidence intervals; this was calculated using the EpiR library of the R program. Among the confirmed COVID-19 patients, we identified the sensitivity of RAT according to various E gene Ct cutoff values of RT–PCR. On admission, the sensitivity of RAT was investigated when each Ct value interval of the E gene was defined for COVID-19-positive patients. Statistical significance was determined when the We included 1875 patients who had RAT and RT–PCR concurrently and who visited the emergency department. The baseline characteristics of the study population are summarized in p value < 0.001), suggesting a high viral load , 62.6–72.7); the specificity was 99.9%  . The numral load .We examined the sensitivity of RAT according to the E gene Ct cutoff value of RT–PCR in COVID-19 patients . When thIn the current study, we aimed to investigate the diagnostic performance of the SAR-CoV-2 Ag test as a screening tool in the ED. The RAT showed high specificity, PPV and NPV, and low overall sensitivity (67.8%). The sensitivity was strongly correlated with the Ct value and ranged from 14.3% (Ct > 30.0) to 97.8% (Ct ≤ 15.0).The NAATs and the antigen test are used as viral tests to detect SAR-CoV-2 infection and as screening tests to decrease the spread of SAR-CoV-2 by identifying and isolating infected individuals . NAATs aAccording to the current guidance from the World Health Organization, the RAT can offer a faster and less expensive method to diagnose active SARS-CoV-2 infection than NAATs . The diaThe Ct value could be used as a marker for predicting the contagion and viral load of an individual, and a significant relationship between a low Ct value and a high risk of secondary transmission was reported . AdditioThe length of stay (LOS) and crowding are significantly associated with the outcomes of patients in the ED ,28. DuriThe current study has several limitations. First, the present study was conducted in a single center, and the study had a retrospective design. These factors could affect the results of the study, and if this study had included individuals from countries with different healthcare systems and races, the results may have been different. Second, we performed the RAT with one product . The sensitivity and specificity could differ when using other RAT commercial products. Third, although we tried to analyze variables, there could be hidden confounders that could affect the results. Fourth, RT–PCR was conducted within 48 h in all included individuals; however, the time gap between RT–PCR and RAT was not analyzed. Fifth, during the study period of the current study, the COVID-19 community level was extremely high. This could affect the results of the study.The sensitivity of RAT could be considered high in patients with a high viral load. The RAT could be used as a screening tool in the ED, especially when the number of COVID-19 patients is high in the community. Future prospective studies are needed to enhance the findings of the current study.